eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 22

also eliminates the very transient background
fluorescence generated from sample components
such as buffers, proteins, and chemicals, which
hinders classic FRET methods.
Ideal
fluorophores have high signal
ratios.
Fluorophores
intensity,
are highly stable, and offer excellent signalto-noise
commonly
used
are " Lanthanide probes " which are metal ions
referring to elements Cerium to Lutetium in
the periodic table. The most used are Europium
and Terbium, which fluoresce over milliseconds
instead of nanoseconds. In practice, a comparison
measurement of the two emitted wavelengths
over time is calculated for a TR-FRET response.
Monitoring BCL-2 Binding to Ligand
BCL-2 (B-cell lymphoma 2), a member of the
BCL family, is an integral protein of the outer
mitochondrial membrane. BCL-2 family members
form hetero- or homodimers that regulate
apoptosis. The main function of BCL-2 is to inhibit
apoptosis and promote cell survival through
control of mitochondrial membrane permeability,
blocking the release
of
cytochrome c from
mitochondria by pro-apoptotic BH3-containing
proteins, and inhibition of caspase activity.
However, BCL-2 may either promote or suppress
apoptosis depending on context and partners.
Constitutive expression of BCL-2 in B lymphocytes
caused by translocation of the BCL-2 gene to Ig
heavy chain locus promotes follicular lymphoma.
The protein contributes to cancer resistance to
treatment in leukemia, melanoma, and breast and
prostate cancer, owing to its pro-survival effects.
Inhibitors used in the clinic are BH3-mimetic
molecules that prevent BCL-2 interaction with its
BH3-type partners. Navitoclax (ABT-263) inhibits
BCL-2, BCL-xL, and BCL-w, while venetoclax (ABT199)
is highly selective of BCL-2.
Figure 3, Left panel: Illustration of the assay principle (BPS Bioscience #50222). Right: Increasing concentrations of ABT199 were added
to a terbium-labeled donor, a dye-labeled acceptor, purified His-tagged BCL-2 protein, peptide ligand, and incubated for 3 hours before
TR-FRET reading. Two sequential measurements were performed: Tb-donor emission was measured at 620 nm followed by dye-acceptor
emission at 665 nm.
22

eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development

Table of Contents for the Digital Edition of eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development

eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 1
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 2
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 3
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 4
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 5
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 6
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 7
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 8
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 9
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 10
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 11
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 12
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 13
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 14
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 15
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 16
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 17
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 18
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 19
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 20
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 21
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 22
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 23
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 24
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 25
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 26
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 27
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 28
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 29
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 30
eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 31
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eBook: Biochemical and Cell-Based Assays for Targeted Cancer Drug Discovery and Development - 33
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